Inflammation is normally an acute response by the immune system to invasion by microbial pathogens, chemicals or physical injury. In some cases, however, the inflammatory response can progress to a chronic state and be the cause of inflammatory disease.
Therapeutic control of this chronic inflammation in diverse diseases is a major medical need. Leukotriene B4 (LTB4) is a potent pro-inflammatory activator of inflammatory cells, including neutrophils (J. Palmblad, J. Rheumatol. 1984, 13(2):163-172), eosinophils (A. M. Tager, et al., J. Exp. Med. 2000, 192(3):439-446), monocytes (N. Dugas et al., Immunol. 1996, 88(3):384-388), macrophages (L. Gagnon et al., Agents Actions 1989, 34(1-2):172-174), T cells (H. Morita et al., Biochem. Biophys. Res. Commun. 1999, 264(2):321-326) and B cells (B. Dugas et al., J. Immunol. 1990, 145(10):3405-3411). Immune cell priming and activation by LTB4 can promote chemotaxis, adhesion, free radical release, degranulation and cytokine release. LTB4 stimulates T-cell proliferation and cytokine release in response to IL-2, concanavalin-A and CD3 ligation (H. Morita et al., Biochem. Biophys. Res. Commun. 1999, 264(2):321-326). LTB4 is a chemoattractant for T-cells creating a functional link between early innate and late adaptive immune responses to inflammation (K. Goodarzi, et al., Nat. Immunol. 2003, 4:965-973; V. L. Ott, et al., Nat. Immunol. 2003, 4:974-981; A. M. Tager, et al., Nat. Immunol. 2003, 4:982-990). There is substantial evidence that LTB4 plays a significant role in the amplification of many inflammatory disease states (R. A. Lewis et al., N. Engl. J. Med. 1990, 323:645; W. R. Henderson, Ann. Intern. Med. 1994, 121:684) including asthma (D. A. Munafo et al., J. Clin. Invest. 1994, 93(3):1042-1050), inflammatory bowel disease (IBD) (P. Sharon and W. F. Stenson, Gastroenterology 1984, 86(3):453-460), chronic obstructive pulmonary disease (COPD) (P. J. Barnes, Respiration 2001, 68(5):441-448), arthritis (R. J. Griffiths et al., Proc. Natl. Acad. Sci. U.S.A. 1995, 92(2):517-521; F. Tsuji et al., Life Sci. 1998 64(3):L51-L56), psoriasis (K. Ikai, J. Dermatol. Sci. 1999, 21(3):135-146; Y. I. Zhu and M. J. Stiller, Skin Pharmacol. Appl. Skin Physiol. 2000, 13(5):235-245), and atherosclerosis (E. B. Friedrich, et al., Arterioscler. Thromb. Vasc. Biol. 2003, 23:1761-1767; K. Subbarao, et al., Arterioscler. Thromb. Vasc. Biol. 2004, 24:369-375; A. Helgadottir, et al., Nat. Genet. 2004, 36:233-239; V. R. Jala, et al., Trends in Immun. 2004, 25:315-322). LTB4 also simulates the production of various cytokines and may play a role in immunoregulation (A. W. Ford-Hutchinson, Immunology 1990, 10:1). Furthermore, it has recently been shown that LTB4 levels are elevated in brochoalveolar lavage fluid from patients with scleroderma lung disease (see Kowal-Bielecka, O. et al., Arthritis Rheum. (Nov. 30, 2005), Vol. 52, No. 12, pp. 3783-3791). Therefore, a therapeutic agent that inhibits the biosynthesis of LTB4 or the response of cells to LTB4 may be useful for the treatment of these inflammatory conditions.
The biosynthesis of LTB4 from arachidonic acid (AA) involves the action of three enzymes: phospholipase A2 (PLA2), to release AA from the membrane lipids; 5-lipoxygenase (5-LO), to form the unstable epoxide Leukotriene A4 (LTA4); and leukotriene A4 hydrolase (LTA4-h), to form LTB4 (A. W. Ford-Hutchinson, et al., Annu. Rev. Biochem. 1994, 63:383-347). The cysteinyl leukotrienes are formed by the addition of glutathione to LTA4 by the action of LTC4 synthase (Aharony, D., Am. J. Respir. Crit. Care Med. 1998, 157 (6, Pt 2), S214-S218) into the pro-inflammatory cysteinyl leukotrienes LTC4, LTD4 and LTE4. An alternative path for LTA4 is conversion via transcellular biosynthesis and the action of lipoxygenases into lipoxin A, (LXA4) and lipoxin B4 (LXB4) (C. N. Serhan, Prostaglandins 1997, 53:107-137).
LTA4-h is a monomeric, soluble 69 kD zinc metalloenzyme. A high resolution crystal structure of recombinant LTA4-h with bound inhibitors has been obtained (M. M. Thunissen et al., Nat Struct. Biol. 2001, 8(2): 131-135). LTA4-h is a bifunctional zinc-dependent metalloenzyme of the M1 class of metallohydrolases. It catalyses two reactions: the stereospecific epoxide hydrolase reaction to convert LTA4 to LTB4 and a peptidase cleavage of chromogenic substrates. The Zn center is critical to both activities. LTA4-h is related to aminopeptidases M and B, which have no LTA4-hydrolase activity. LTA4-h has high substrate specificity, accepting only a 5,6-trans-epoxide with a free carboxylic acid at C-1 of the fatty acid. The double-bond geometry of the substrate is essential for catalysis. LTA3 and LTA5 are the only other weak substrates known to date. In contrast, LTA4-h peptidase activity appears to be promiscuous, cleaving nitroanilide and 2-naphthylamide derivatives of various amino acids, e.g. in particular alanine and arginine. Arg-Gly-Asp, Arg-Gly-Gly, and Arg-His-Phe tripeptides are hydrolyzed with specificity constants (kcat/Km) similar to the epoxide hydrolase reaction. There is no known physiological peptide substrate for LTA4-h.
LTA4-h is widely expressed as a soluble intracellular enzyme in intestine, spleen, lung and kidney. High activity levels are found in neutrophils, monocytes, lymphocytes and erythrocytes. Tissue macrophages can have high LTA4-h levels. An interesting feature is that the cellular distribution of LTA4-h and 5-LO are distinct, requiring close apposition of cells such as neutrophils and epithelial cells for efficient transcellular LTB4 synthesis. Many studies support this concept, including recent data from bone marrow chimeras derived from LTA4-h−/− and 5-LO−/− mice (J. E. Fabre et al., J. Clin. Invest. 2002, 109(10):1373-1380).
These important functions of LTB4 in inflammation and potentially in autoimmunity prompted an aggressive search at numerous pharmaceutical companies to discover potent LTB4 receptor antagonists. These efforts were initiated long before the molecular identity of LTB4 receptors was known. Drug discovery efforts focused on competition binding of small molecule antagonists or agonists at [3H]-LTB4 binding sites and functional responses, e.g. chemotaxis in human neutrophils. Despite the presence of a stereospecific, high affinity [3H]-LTB4 receptor (Kd<1 nM) on human neutrophils, it was apparent from early studies that additional lower affinity LTB4 receptors (Kd>60 nM) were also present on neutrophils (D. W. Goldman and E. J. Goetzl, J. Exp. Med. 1984 159(4):1027-1041). This LTB4 receptor heterogeneity was subsequently confirmed in HL-60 leukemia cells (C. W. Benjamin et al., J. Biol. Chem. 1985, 260(26):14208-14213), alveolar macrophages (A. J. de Brum et al., Prostaglandins 1990, 40(5):515-527), peritoneal eosinophils (R. Sehmi et al., Immunol. 1992, 77(1):129-135) and other cell types.
The seminal work of Takao Shimizu and colleagues in cloning human LTB4 receptors has recently defined two pharmacologically distinct receptors (T. Shimizu et al., Ernst Schering Res. Found. Workshop 2000, (31):125-141). Human BLT1 and its mouse, rat and guinea pig orthologues represent the high affinity LTB4 receptor (Kd 0.1-0.7 nM). BLT1 has a restricted expression in inflammatory cells, e.g. neutrophils, monocytes, thymus and spleen. Human and mouse BLT2 have a wider tissue expression profile than BLT1, with evidence for mRNA transcripts predominantly in spleen, liver, ovary and leukocytes and lower transcript levels in many other tissues (T. Yokomizo et al., J. Exp. Med. 2000, 192(3):421-432; T. Yokomizo et al., J. Biol. Chem. 2001, 276(15):12454-12459). Human BLT2 had 20-fold lower affinity for LTB4 (Kd=23 nM) than BLT1 and much weaker, but measurable affinity for other eicosanoids. The distinct pharmacology of BLT1 and BLT2 receptors was shown by [3H]-LTB4 competition binding studies with industry-standard LTB4 receptor antagonists. Most known LTB4 receptor antagonists were able to compete for binding to BLT1 but not to BLT2.
These findings suggest that local concentrations of LTB4 generated at sites of inflammation will provide graded responses to different cell types based on either unique or regulated co-expression of BLT1 and BLT2 receptors. This was confirmed by co-expression of BLT1 and BLT2 in CHO cells, which exhibited a broader dose response range to LTB4-stimulated chemotaxis than either receptor alone (T. Yokomizo et al., Life Sci. 2001, 68(19-20):2207-2212). The data also suggest that the failure or success of a given LTB4 receptor antagonist in pre-clinical efficacy models of inflammatory or autoimmune disease and in human clinical trials needs to be re-examined in light of pharmacological effects at these distinct BLT1 and BLT2 receptors.
Further analysis of LTB4 receptor subtype expression in immune cells has been performed by semi-quantitative PCR analysis (T. Yokomizo et al., Life Sci. 2001, 68(19-20):2207-2212). Data suggest BLT1 mRNA expression is highest in CD14+ monocytes, while BLT2 mRNA expression is high in CD8+ cytotoxic T-, CD4+ helper T-, and CD19+ B-cells. These findings have not been corroborated with clear evidence for differential BLT1 and BLT2 expression at the protein level. Although a BLT1-specific antibody has been reported (A. Pettersson et al., Biochem. Biophys. Res. Commun. 2000, 279(2):520-525), anti-BLT2 antibody are not yet available. Nevertheless, the known responses of some of these cell types to LTB (see above) suggest a role for BLT2 in modulating T- and B-lymphocyte-dependent immune biology. While an LTB4 receptor antagonist may differ in its affinity for BLT1 vs BLT2, blocking the production of LTB4 using LTA4-h inhibitors would be expected to inhibit the downstream events mediated through both BLT1 and BLT2.
Studies have shown that introduction of exogenous LTB4 into normal tissues can induce inflammatory symptoms (R. D. R. Camp et al., Br. J. Pharmacol. 1983, 80(3):497-502; R. Camp et al., J. Invest. Dermatol. 1984, 82(2):202-204). Elevated levels of LTB4 have been observed in a number of inflammatory diseases including inflammatory bowel disease (IBD), chronic obstructed pulmonary disease (COPD), psoriasis, rheumatoid arthritis (RA), cystic fibrosis, multiple sclerosis (MS), and asthma (S. W. Crooks and R. S. Stockley, Int. J. Biochem. Cell Biol. 1998, 30(2):173-178). Therefore, reduction of LTB4 production by an inhibitor of LTA4-h activity would be predicted to have therapeutic potential in a wide range of diseases.
This idea is supported by a study of LTA4-h-deficient mice that, while otherwise healthy, exhibited markedly decreased neutrophil influx in arachidonic acid-induced ear inflammation and zymosan-induced peritonitis models (R. S. Byrum et al., J. Immunol. 1999, 163(12):6810-68129). LTA4-h inhibitors have been shown to be effective anti-inflammatory agents in preclinical studies. For example, oral administration of LTA4-h inhibitor SC57461 caused inhibition of ionophore-induced LTB4 production in mouse blood ex vivo, and in rat peritoneum in vivo (J. K. Kachur et al., J. Pharm. Exp. Thr. 2002, 300(2): 583-587). Eight weeks of treatment with the same inhibitor significantly improved colitis symptoms in cotton top tamarins (T. D. Penning, Curr. Pharm. Des. 2001, 7(3):163-179). The spontaneous colitis that develops in these animals is very similar to human IBD. The results therefore indicate that LTA4-h inhibitors would have therapeutic utility in this and other human inflammatory diseases.
Inflammation may be observed in any one of a plurality of conditions, such as asthma, COPD, atherosclerosis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel diseases (IBD, including Crohn's disease and ulcerative colitis), or psoriasis, which are each characterized by excessive or prolonged inflammation at some stage of the disease. The connection between inflammatory diseases and cancer has been strengthened by the strong link established between a mutation of the oncogene ras and a de-novo expression of the BLT2 receptor as well as activation of LTB4 synthesis in tumor cells (M.-H. Yoo et al. 2004, Oncogene, 23, 9259). Previously it was shown in various cell models that oncogenic ras induces cytosolic phospholipase A (cPLA2) thus increasing the release of arachidonic acid (L. E. Heasley et al. 1997, J. Biol. Chem., 272, 14501) and the synthesis of LTB4. Inhibition of this pathway through an LTA4-h inhibitor would have a therapeutic utility in the treatment of cancers.
Events that elicit the inflammatory response include the formation of the pro-inflammatory mediator LTB4, which can be blocked with an LTA4-h inhibitor, thus providing the ability to prevent and/or treat leukotriene-mediated conditions, such as inflammation. The inflammatory response is characterized by pain, increased temperature, redness, swelling, or reduced function, or by a combination of two or more of these symptoms. Regarding the onset and evolution of inflammation, inflammatory diseases or inflammation-mediated diseases or conditions include, but are not limited to, acute inflammation, allergic inflammation, and chronic inflammation.
Background and review material on inflammation and conditions related with inflammation can be found in articles such as the following: C. Nathan, Points of control in inflammation, Nature 2002, 420:846-852; K. J. Tracey, The inflammatory reflex, Nature 2002, 420:853-859; L. M. Coussens and Z. Werb, Inflammation and cancer, Nature 2002, 420:860-867; P. Libby, Inflammation in atherosclerosis, Nature 2002, 420:868-874; C. Benoist and D. Mathis, Mast cells in autoimmune disease, Nature 2002, 420:875-878; H. L. Weiner and D. J. Selkoe, Inflammation and therapeutic vaccination in CNS diseases, Nature 2002, 420:879-884; J. Cohen, The immunopathogenesis of sepsis, Nature 2002, 420:885-891; D. Steinberg, Atherogenesis in perspective: Hypercholesterolemia and inflammation as partners in crime, Nature Medicine 2002, 8(11):1211-1217. Cited references are incorporated herein by reference.
The connection between members of the leukotriene pathway, particularly LTA4-h and LTB4, and myocardial infarction and acute coronary syndrome has recently been disclosed in PCT Published Patent Application WO 2004/035741, PCT Published Patent Application WO 2004/035746, PCT Published Patent Application WO 2005/027886, PCT Published Patent Application WO 2005/075022, and U.S. Published Patent Application US 2005/0113408, the pertinent disclosures of which are incorporated by reference in their entireties, and in Nature Genetics, Advanced Online Communication, Nov. 10, 2005.
Accordingly, there exists a need for inhibitors of the LTA4-h enzyme, particularly inhibitors that are useful in the inhibition of pro-inflammatory mediators, such as the LTB4 mediator. Such inhibitors would be useful in the treatment of diseases and conditions as set forth herein.